This goal of the project is the study of the biology of cartilage tissues and the application of such knowledge to musculoskeletal and orthopaedic medicine. This project focuses on cartilage development, functional cartilage tissue engineering, the cellular basis of orthopaedic implant stability, physical influences on skeletal tissue function, and the biology of skeletal tissue injury repair. [unreadable] [unreadable] There are six interrelated parts to the project:[unreadable] [unreadable] (1) Cellular and Molecular Mechanisms of Mesenchymal Chondrogenesis. Building on our previous investigations on embryonic limb cartilage development, we have focused our studies on the mechanisms responsible for chondrogenesis of adult mesenchymal stem cells, which represent a highly promising candidate cell type for cartilage tissue engineering and regeneration. Multipotent mesenchymal stem cells are isolated from bone marrow stroma and from adult trabecular bone and studied in vitro for their ability to undergo multi-lineage differentiation along the osteogenic, chondrogenic, and adipogenic pathways. The mechanisms of action of growth factors (e.g. TGF-beta superfamily members), signaling molecules (e.g. Wnts), hormonal regulators (e.g. glucocorticoids), as well as small molecules (e.g., glucosamine) in the maintenance of their undifferentiated state and lineage commitment are being analyzed. Results point to the correlation between developmental and regenerative tissue morphogenesis, and suggest that these signaling molecules and their down-stream signal mediators are potential targets for gene-based modulation of adult tissue genesis using mesenchymal stem cells. Novel methods have been developed for efficient gene transduction, using electrical field based and nucleofection protocols, to modulate the expression of these key factors and related signaling pathways in mesenchymal stem cells and to examine the effects on cellular differentiation. [unreadable] [unreadable] (2) Mesenchymal Stem Cells and Stemness Genes. We are also currently developing clonal mesenchymal stem cell lines harboring differentiation-specific marker gene constructs as read-out cell systems, e.g. for functional gene cloning applications, as well as gene microarray approaches to profile gene expression profiles during differentiation to identify key regulatory signals. Particularly noteworthy is our recent finding that mesenchymal stem cells possess transdifferentiation potential, underscoring the possibility of identifying stemness regulatory genes.&#8232;&#8232; Using these genes as well as re-programming genes recently identified as active in embryonic stem cells, we are exploring the feasibility of maintenance or restoration of stemness to mesenchymal stem cells that have senesce in vitro. In addition, we are exploring the nature of the mesenchymal stem cell niche by analyzing the interaction between differentiated cell types that are adjacent to stem cells in vivo (e.g., enthothelial cells and osteoblasts) and mesenchymal stem cells. Our results show that the interactions affect the cell fate of both stem cells and the differentiated cells, suggesting these interactions may contribute towards the stem cell niche in vivo.[unreadable] [unreadable] (3) Cell- and Biomaterial-Based Cartilage Tissue Engineering. We are developing new methods to construct three-dimensional biodegradable scaffolds to seed Mesenchymal stem cells under chondrogenic conditions ex vivo for cartilage tissue engineering applications. Specifically, our efforts are targeted towards articular cartilage of the joint, and fibrocartilage of the meniscus. Current work is focused on the following: (a) development of novel three-dimensional nanofibrous scaffolds consisting of biodegradable polymers produced by electrospinning, and subsequent alignment of the fibrous network; (b) application of hydrogels, such as agarose and collagen to encase Mesenchymal stem cells and chondrocytes, for subsequent mechano-stimulation of tissue formation and cell-cell interaction; (c) custom-fabrication of biomaterial scaffolds to assemble cartilage tissue in custom-designed molds that retain shape memory; (d) development of an articular cartilage degeneration model (rabbit) initiated by supraphysiological impact; (e) in vivo testing of the applicability of tissue-engineered cartilage for articular cartilage repair in a rabbit model; and (f) application of biomaterials for controlled release and delivery of bioactive factors and genetic materials to promote cell differentiation and tissue formation.[unreadable] [unreadable] (4) Cellular Mechanism of Wear Debris Mediated Osteolysis. Our recent studies have also addressed the cellular mechanisms responsible for implant wear debris mediated osteolysis, which is primarily responsible for aseptic implant loosening. Specifically, our results indicate that the presence of titanium particulate debris suppresses osteogenic differentiation and enhances apoptosis in cultures of Mesenchymal stem cells in vitro, both of these responses likely contributing to compromised periprosthetic osteogenic tissue response and implant loosening. The identity and involvement of specific cytokines and signaling pathways in the affected cellular events are being investigated, using ex vivo approaches, including testing a tissue analogue to mimic bone-implant interaction in vitro. [unreadable] [unreadable] (5) Analysis of Physical Influences on Skeletal Biology. Skeletal tissues are uniquely adapted to responding to mechanical influences. We are currently designing mechanoactive bioreactor systems to apply both dynamic and static mechanical stimulation based on hydrostatic and compressive loading, and using mesenchymal stem cell-based tissue constructs to analyze the cellular and molecular basis of the biological responses. By varying the nature of the biomaterial scaffold, the mixture of cell types, and the growth factor treatment, we aim to decipher the crosstalk among various signal transduction pathways. More importantly, we are examining the interaction between different cell types under mechanoactive environment. We are also applying a mechanoactive bioreactor system for tendon/ligament tissue engineering. [unreadable] [unreadable] (6) Animal Models of Skeletal Injury Repair and Regeneration. Two mouse models are being investigated: (a) the role of GDF5 in mouse tibial fracture repair, and (b) cellular and molecular analysis of distraction osteogenesis in a mouse model. [unreadable] [unreadable] [unreadable] Information gathered from this project should provide a rational basis to functional skeletal tissue engineering.